Von Willebrand Factor (VWF) and Factor VIII (FVIII) are key central proteins in the initiation and regulation of[unreadable] hemostasis. Each has its own hereditary deficiency disease, which cause either VWD or hemophilia A, and they[unreadable] circulate together as a non-covalent complex in plasma. This project explores the requirements for the establishment[unreadable] of a regulated secretory pathway for one or both VWF and FVIII proteins within endothelial cells and[unreadable] megakaryocytes. Aim 1 will determine the structural and functional requirements within VWF for the storage[unreadable] of both VWF and FVIII and contrasts these pathways within endothelial cells and platelets. The Weibel-Palade[unreadable] body has an absolute requirement for synthesis of pro-VWF while the alpha granule is permissive if pro-VWF is[unreadable] synthesized. Specific Aim 2 will determine the in vivo potential for synthesis of FVIII in the presence of VWF[unreadable] as a novel therapeutic approach in the treatment of hemophilia A. Transgenic (F8-/-) animals expressing FVIII[unreadable] only in either endothelium or megakaryocytes will be studied to determine the therapeutic advantage of expressing[unreadable] FVIII with VWF. Specific Aim 3 will examine the potential for platelet-specific or endothelial cell-specific[unreadable] FVIII expression as a means to bypass the inhibitory activity of FVIII antibodies. Transgenic animals[unreadable] expressing FVIII in endothelial cells or platelets will be studied within the context of inhibitory antibodies to[unreadable] determine if these sites of expression can "bypass" plasma inhibitors by locally releasing active FVIII in the context[unreadable] of VWF. Specific Aim 4 will determine if the endothelial cell is the physiologic site for expression of FVIII or[unreadable] at least the site synthesizing and storing the FVIII released by the administration of DDAVP. Since the[unreadable] controversy continues as to the site of normal FVIII synthesis, these studies will explore whether the DDAVP[unreadable] releasable pool of FVIII has an absolute requirement for endothelial synthesis of FVIII or whether it can be created in[unreadable] the absence of specific endothelial cell synthesis. Tissue specific knockouts of FVIII production will be carried out[unreadable] in the hepatocyte or in the endothelial cell to determine if these tissue-specific knockouts caused a marked reduction[unreadable] in plasma FVIII. Therefore, we feel that tiiese aims will definitively explore the function of VWF on the in vivo and[unreadable] in vitro intracellular biology of FVIII and provide potential for new therapeutic approaches to the treatment of[unreadable] hemophilia A both in patients with and those without FVIII inhibitors.